Both the actin cytoskeleton and cholesterol-rich liquid-ordered membrane domains, also called 'lipid rafts', are involved in signal transduction during cell adhesion, motility, survival, and membrane trafficking. However, the underlying molecular mechanisms are unclear. This application proposes the continued study of a new type of membrane skeleton, first described and characterized in the current funding period, that may represent a 'missing link'in understanding actin and myosin involvement in lipid raft signaling. Detergent-resistant membranes containing this membrane skeleton (DRM-H) exhibit a higher buoyant density than do other cholesterol-rich membrane fractions due to the tight association of lipid raft organizing proteins (flotillins, stomatin) and signaling proteins (Src family kinases, heterotrimeric Gi proteins, matrix metalloproteinase) with filamentous actin, myosins I and II, and other membrane skeleton proteins (alpha-actinin, fodrin, supervillin). DRM-H-related membrane skeletons are present in many motile cells and apparently regulate cell adhesion, contractility, and signaling to extracellularly regulated kinases (ERK1/2). We propose that the DRM-H membrane skeleton is involved in Src and/or ERK signaling by promoting local rearrangements of lipid raft-associated proteins. Supervillin, a membrane-proximal protein that also binds directly to actin and myosin II, appears to be a key control point for this regulation. To test these hypotheses, we propose to: (1) elucidate the roles of actin, myosin, supervillin, and other DRM-H proteins in the formation and reorganization of signaling scaffolds;(2) explore the mechanisms by which DRM-H proteins modulate focal adhesion function and cellular contractility;and (3) determine the role of the DRM-H membrane skeleton during membrane trafficking. These studies describe a novel mechanism for the attachment of actin and myosin II to cholesterol-rich membrane domains and will provide insight into cytoskeletal regulatory mechanisms in these domains. The long-range goal of this project is to understand how actin-based membrane skeletons function during cell motility, adhesion, and signaling. This information will increase our understanding of both normal cellular behaviors and pathological conditions associated with immune dysfunction, cancer cell invasion, muscular dystrophy, diabetes, and developmental abnormalities.